Analysis of unknown proteins is a very important technical ability in biochemistry, and has many applications in the fast growing biochemical related industries. There are a number of different techniques that have been developed and apparatus designed to facilitate the procedures. As an example, preparative gel electrophoresis apparatus has been developed and automated for separating macromolecules, such as proteins, in a sample mixture of unknown materials.
After isolating a protein, it is often desirable to break the strong peptide bonds by which the constituent amino acids are chained, and to tag and quantify the individual amino acids to help identify the protein. It is in this latter general procedure that the present invention has most utility.
One method by which peptide bonds may be broken is through hydrolysis by the action of a strong mineral acid, such as hydrochloric acid (HC1). In a manual laboratory technique, a number of isolated protein samples may be placed in individual containers, such as test tubes, and the tubes then placed upright in a larger container, such as a beaker, which will completely enclose the separate tubes. The upright tubes containing proteins are left open to the inside of the beaker. A concentrated solution of an acid, typically HC1, is placed in the bottom of the beaker, not in direct contact with any of the proteins. The top of the beaker is closed by a suitable method, and heat is applied, causing the HC1 solution to form a vapor in the closed beaker. The vapor contacts the protein sample in each test tube, and over a period of time, the peptide bonds are broken, reducing each protein sample to its constituent amino acids. After the hydrolysis, each of the samples may be introduced by suitable techniques for chromatographic analysis.
The hydrolysis process requires the addition of heat, and intimate contact between the protein and the vapor. The process is also rather slow, and a sample may require typically 20 hours or more at process conditions for the complete dissolution of the bonds in a protein sample. One of the reasons the process takes a lot of time is the limited area of contact between the HC1 and the protein sample. If the dissolution is not complete, the analysis may be faulty.
Another difficulty in this kind of analysis, as in others, is a requirement for clean apparatus and technique so that one sample is not contaminated by another, causing error in the analysis. In the manual procedure, processing more than one sample at a time accepts a risk of cross contamination between samples. Also, there is often a loss of sample in the removal from the beaker after hydrolysis. Moreover, the apparatus must be thoroughly and manually cleaned between procedures.
After hydrolysis is performed, it is still necessary that each sample be tagged, which is a process of exposing the sample to a tagging solution such as phenylisothiocyanate, often with mild heating. Then the samples are introduced for chromatographic analysis, where UV absorption techniques produce measurements, which, by comparison with results from standard samples, may be used to determine the amino acid composition of a protein.
The overall sequence of processes from protein preparation to identifying chromatogram is time consuming, subject to considerable human and equipment error, and open to contamination at several stages. Control is uncertain. What is clearly needed is an automated apparatus in which hydrolysis and derivatization of protein samples may be quickly and efficiently accomplished under programmed control. Such apparatus needs to maximize reaction velocity, provide for simple and efficient automatic cleaning, perform all needed steps without manual intervention, and provide for operation with a large number of samples.